The scheme, and regulative mechanism of pyroptosis, ferroptosis, and necroptosis in radiation injury

Radiotherapy (RT) stands as the primary treatment for tumors, but it inevitably causes damage to normal cells. Consequently, radiation injury is a crucial consideration for radiation oncologists during therapy planning. Cell death including apoptosis, autophagy, pyroptosis, ferroptosis, and necroptosis play significant roles in tumor treatment. While previous studies elucidated the induction of apoptosis and autophagy by ionizing radiation (IR), recent attention has shifted to pyroptosis, ferroptosis, and necroptosis, revealing their effects induced by IR. This review aims to summarize the strategies employed by IR, either alone or in combination therapy, to induce pyroptosis, ferroptosis, and necroptosis in radiation injury. Furthermore, we explore their effects and molecular pathways, shedding light on their roles in radiation injury. Finally, we summarize the regulative agents for these three types of cell death and their mechanisms. In summary, optimizing radiation dose, dose rate, and combined treatment plans to minimize radiation damage and enhance the killing effect of RT is a key focus.


Review
The scheme, and regulative mechanism of pyroptosis, ferroptosis, and necroptosis in radiation injury

Introduction
Annually, approximately 470,000 patients undergo radiotherapy (RT) in the United States, with up to half of all cancer patients worldwide anticipated to receive this treatment [1].When integrated with surgery [2], chemotherapy [3], targeted therapy [4], immunotherapy [5], and other modalities, RT proves effective in inhibiting tumor progression, extending patient survival, and enhancing the quality of life.Mechanistically, the traditional perspective suggests that RT directly affects biological macromolecules such as DNA and proteins [6].It also indirectly impacts water molecules, generating radiolysis products that cause DNA damage and protein denaturation, ultimately resulting in tumor cell death [7].Furthermore, with the advent of immunotherapy, the role of the immune system in tumor treatment has gained attention.Numerous studies have demonstrated that RT functions as an "in situ vaccine", activating the immune system and promoting anti-tumor immunity [8].
Cell death can be categorized into nonprogrammed cell death, and programmed cell death [33,34].While apoptosis has traditionally been regarded as the primary mechanism of cell death, the recognition of alternative forms, such as programmed necrosis, has challenged this conventional perspective [35,36].Pyroptosis [37,38], ferroptosis [39,40], and necroptosis [41,42], which have gained significant attention in recent years, fall under the category of programmed cell necrosis.
Ongoing research on cell death induced by IR, leading to either tumor cell elimination or normal tissue damage [32].The cell death induced by IR extends beyond apoptosis [70] and autophagy [71].It exerts its effects by triggering a variety of cell death pathways, including pyroptosis, ferroptosis, and necroptosis.Recently, radiobiologists have initiated investigation into the roles of radiation-induced pyroptosis, ferroptosis, and necroptosis.This review aims to understand the protocols and molecular regulatory pathways involved in radiation damage mediated by pyroptosis, ferroptosis, and necroptosis, both in the context of RT alone or in combination with other treatments.
The occurrence of pyroptosis, ferroptosis and necroptosis induced by IR is dependent on the dose and dose rate.Generally, the total IR dose for both pyroptosis, ferroptosis and necroptosis is below 10 Gy, occasionally ranging from 10-25 Gy.Notably, Wang et al. employed a higher dose of 40 Gy to induce ferroptosis, establishing a radiation-induced skin injury model in Sprague-Dawley (SD) rats [93].Compared with ferroptosis and necroptosis, pyroptosis requires a larger total IR dose.For ferroptosis and necroptosis, the administered dose to animals is relatively higher than that given to cells, with no obvious trend observed in pyroptosis (Figure 1) (Supplementary Table 1).Regarding dose rates, 1-4 Gy/min is commonly applied.Zhang et al. conducted a study using a high dose rate of 12.61 Gy/min to induce pyroptosis [96].No distinct pattern emerges between cell death modes and cell/animal models.However, parameters such as total dose, fractionation, and dose rate should be considered when constructing a radiation injury model.Additionally, animal factors, including the use of C57BL/6J mice, ICR mice, SD rats, and Balb/c mice, should be taken into account.Due to variations in radiation sensitivities, the IR parameters used to induce damage in different organs vary.The doses for radiation injury induction are often higher than those for sensitivity enhancement.In animal models, there is no clear pattern regarding the radiation dose inducing pyroptosis, ferroptosis or necroptosis, and the type of cell death produced by similar doses is uncertain.Common parameters of radiation injury caused by pyroptosis, ferroptosis and necroptosis are presented in Supplementary Table 1.

Various
agents, including biological products/extracts, nuclear factor kappa-B (NF-κB) inhibitors, and different compounds, can inhibit radiation-induced pyroptosis and mitigate radiation injury.In Supplementary Table 2, we have compiled a summary of pyroptosis inhibitors that provide protection against radiation injury, along with elucidation of their underlying mechanisms.Plant extracts or microbial products have shown inhibitory effects on pyroptosis, functioning as radiation protection factors.For instance, ACT001, derived from parthenolide found in the feverfew plant, inhibits NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and reduces the expression of IL-1β, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and GSDMD.This ultimately inhibits pyroptosis and radiation-induced lung injury (Figure 2A) [73].Andrographis paniculate extract and its active compound andrographolide, along with the traditional Chinese medicine Re-Du-Ning (RDN) demonstrate pronounced efficacy in preventing absent in melanoma 2 (AIM2) from sensing DNA damage caused by IR.These agents effectively pyroptosis-induced pneumonia and suppress epithelial-mesenchymal transition (EMT)mediated pulmonary fibrosis [97,98].Cui et al. discovered that I-Histidine, secreted by intestinal flora, and its metabolite imidazole propionate attenuate radiation toxicity in the heart and lungs by inhibiting pyroptosis (Figure 2B) [99].Additionally, Xu's team confirmed that bacterially derived flagellin A N/C (FlaAN/C) contributes to the restoration of intestinal vitiligo and the reduction of hemorrhage areas by inhibiting pyroptosis and apoptosis.FlaAN/C significantly inhibits ROS, NLRP3, and caspase-1 in intestinal cells post-IR, subsequently diminishing the release of inflammation-related cytokines such as IL-1β, IL-6, IL-18, interleukin-8 (IL-8), and TNF-α (Figure 2C) [80].Biological extracts have emerged as significant agents for suppressing pyroptosis related radiation injury.
The NF-κB pathway has been demonstrated as a target of pyroptosis to govern radiation injury.Alterations in the immune microenvironment pre and post RT contribute to the development of radiation-induced damage [104,105].In contrast to apoptosis, known for immune silencing, pyroptosis disrupts the cell membrane and releases cellular contents, triggering immune activation [41,106].The impact of IR on the immune system has garnered substantial attention [107,108].Han et al. reported that IR induces pyroptosis and recruits cytotoxic T cells through the caspase-9/caspase-3/gasdermin E (GSDME) signaling pathway [109].Combining IR with chemotherapy drugs like cisplatin, etoposide, decitabine, and azacytidine has the potential to further enhance anti-tumor immunity [109].IR-induced pyroptosis can influence radiationinduced damage development by modulating macrophages, dendritic cells, T cells, and natural killer cells (NK cells).Andrographolide (Figure 3Aa) and miR-223-3p (Figure 3Ab) inhibit caspase-1mediated macrophage pyroptosis by targeting the AIM2 and NLRP3 inflammasomes, thereby mitigating radiation-induced lung injury [96,97].The combination of RDN and IR effectively prevents pyroptosis and radiation pneumonitis.Mechanistically, RDN suppresses the activation of the PI3K/AKT pathway and AIM2, blocking immune cells infiltration like macrophages, neutrophils, and T lymphocytes (Figure 3Ac) [98].Compared to conventional RT, FLASH-RT attenuates GSDMEmediated pyroptosis by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway.This reduction in activity leads to a decrease in CD8+ T cells recruitment and subsequently mitigates radiation-induced intestinal damage (Figure 3Ba) [110].Additionally, Wu et al. demonstrated that knocking out cGAS downregulates caspase-11-mediated pyroptosis and reduce sepsis following IR [72].Interestingly, GSDME/caspase3induced pyroptosis and NK cells activation exhibit a dual effect, concurrently diminishing radiation resistance and increasing radiation-related organ toxicity in the intestine, liver, stomach, and pancreas (Figure 3C) [111].Macrophages, T cells, neutrophils, and NK cells within the immune microenvironment play crucial roles in the process of pyroptosis implicated in the development of radiation injury.The relevant molecular mechanisms have been summarized in Supplementary Table 3.
Other substances, such as NaNO3, pulmozyme, and mesenchymal stem cells, show potential as radioprotective agents.NaNO3 has been identified to reduce the radiation-induced expression of GSDMD, GSDMD-NT, ASC, and IL-18, thereby inhibiting NLPR3-mediated pyroptosis and protecting acinar cells from damage (Figure 2J) [76].Pulmozyme, a widely used clinical drug for cystic fibrosis, can inhibit double-strand break of DNA damage induced by IR and the activation of the cGAS/STING/NLRP3 signaling pathway.This inhibition subsequently prevents pyroptosis and radiation-induced lung damage (Figure 2K) [112].Mesenchymal stem cells possess the capability to suppress the activation of NLRP3 and caspase-1, thereby attenuating microglial pyroptosis and protecting brain tissue from radiation injury (Figure 2L) [75].Biological extracts, NF-κB pathway regulators, and compounds with special structures have been revealed to be effective agents targeting pyroptosis to treat radiation injury.
Exosomes and plasmids serve as important carriers for cell-free therapies, gaining notable attention in recent years [117,118].Ferroptosis plays a crucial role in repairing radiation-induced damage to the hematopoietic system and skin.Yao et al. reported that the injecting exosomes derived from healthy rats' plasma (RPExos) promoted fibroblast growth and wound healing in mouse skin injuries caused by irradiation [91].Additionally, plasmid-loaded human MnSOD downregulates acyl-CoA synthetase long-chain family member 4 (ACSL4) and reduces ROS production.It concurrently upregulates the expression of GPX4 and SLC7A11 to attenuate ferroptosis and radiation-induced skin damage [93].Exosomes and plasmids hold promise as important delivery vehicles for targeting ferroptosis in the management of radiation injury.
Melatonin and cholesterol have demonstrated radioprotective potential in preclinical studies (Figure 4).Du's team reported that melatonin, secreted by the pineal gland, promotes the binding of NRF2 to pyruvate kinase isozymes M2 (PKM2) and facilitates its nuclear translocation.This protective mechanism mitigates radiation-induced hippocampal neuron death by attenuating ferroptosis [92].Cholesterol enhances the ferroptosis resistance of bone marrow hematopoietic stem cells (Figure 4).This is achieved through the activation of the solute carrier family 38 member 9 (SLC38A9)/mammalian target of rapamycin (mTOR) axis and SLC7A11/GPX4 axis, along with the inhibition of ferritinophagy.These mechanisms collectively attenuate radiation-induced myelosuppression [119].Conversely, Tyurina et al. reported that pseudomonas aeruginosa (PAO1) induces ferroptosis by promoting the generation of 15-HpETE-PE and oxidized phosphatidylethanolamine through 15-lipoxygenase.This further enhances intestinal damage [83].Human endogenous metabolites could serve as crucial components of radioprotectants by exerting inhibitory effects on ferroptosis.
Emerging evidence indicates a close relationship between radiation-induced ferroptosis and immune system activation.Hu et al. demonstrated that the restoration of radiation-induced intestinal immune imbalance by the ferroptosis inhibitor liproxstatin-1 (Figure 3Bb) [122].Furthermore, ferrostatin-1 (Figure 3Da), LDN 193189 (Figure 3Db), and DFO have been shown to reduce bone marrow suppression and restore the number of red/white blood cells by inhibiting iron metabolism.Consequently, this intervention alleviates acute radiation symptoms such as bleeding and prolongs mouse survival [89,90].Additionally, Ding et al. observed ferroptosis induction in AHH-1 lymphocytes following exposure to low-dose radiation (0-4.7 mGy) [123].However, the expression of ferroptosis markers decreased after 4.8-28.8mGy irradiation.Lymphocytes and monocytes, integral components of the immune microenvironment, have been identified as crucial mediators in the development of radiation injury triggered by ferroptosis.

Regulation of radiation induced-necroptosis and its mechanisms
Plant extracts, such as crocetin derived from gardenia fruit and lipoxygenase-15 derived from baicalein, exhibit potential as radioprotective drugs (Figure 5).Crocetin alleviates radiation injury to lung tissue structure by inhibiting the expression of Tnfrsf10b, thereby hindering the occurrence of necroptosis [94].Greenberger et al. reported that lipoxygenase-15, along with the classic necroptosis inhibitor necrostatin-1 can inhibit necroptosis and extend the survival of radiation-damaged animals [124].Additionally, Kagan's team demonstrated that necrostatin-1 (Figure 5) inhibits necroptosis and counters radiation-induced lethal damage by suppressing the expression of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and phosphorylating RIPK3 [125].Furthermore, NRF2, a key regulator of ferroptosis, has been identified as a suppressor of necroptosis, alleviating radiation-induced rectal injury [95].Agents with the capacity to suppress necroptosis show promise as innovative radioprotective therapies.Necroptosis inhibitors, along with their protective mechanisms against radiation injury are summarized in Supplementary Table 2.

Clinical translations
In recent years, clinical trials studying pyroptosis, ferroptosis and necroptosis have emerged.However, trials specifically targeting pyroptosis, ferroptosis, necroptosis, and radiation injury are rare.
Wang et al. summarized clinical trials focusing on pyroptosis, ferroptosis, and necroptosis in the context of RT [126].Most of these clinical trials are in phase I or II, with few in phase III.Nevertheless, their emphasis lies predominantly on alterations in the expression of key regulatory molecules associated with pyroptosis, ferroptosis, and necroptosis, which may not comprehensively depict the incidence of cell death [126].Presently, clinical trials addressing radiation injury primarily concentrate on radiation dermatitis [127][128][129], with a limited number focusing on radiation pneumonitis [130] and radiation enteritis [131].The majority of interventions remain symptomatic, with approaches like hyperbaric oxygen therapy, administration of glucocorticoid, pain management, and nutritional support [13,14,[132][133][134].The clinical efficacy of therapies targeting cell death for radiation injury treatment is yet to be substantiated.Currently, only a sparse number of clinical trials are investigating drugs with potential cell death inhibitory effects in the context of radiation injury, such as enalapril and captopril [135][136][137][138].The actual impact of these drugs on improving radiation injury through the inhibition of cell death necessitates further confirmation.

Conclusion and prospective
Radiotherapy (RT) is a double-edged sword.While it effectively kills tumor cells, it also damages surrounding normal tissues, causing radiation injury [139,140].Cell death serves as one of the mechanisms that explains both phenomena.This review focuses on the roles of pyroptosis, ferroptosis, and necroptosis in the radiation injury process, attracting significant attention among oncologists.
Overall, the occurrence of pyroptosis, ferroptosis, and necroptosis plays a significant role in mediating radiation injury to a certain extent.Most studies on pyroptosis, ferroptosis, and necroptosis in the field of radiation injury use combined inducers or inhibitors, such as biological extracts and NF-κB inhibitors.Current investigations into radiation injury to normal tissue do not concurrently address the radiation resistance of tumor tissue, deviating from the actual clinical scenario.However, a few studies have indicated that ferroptosis is inhibited under radiation, potentially associated with tumor type and radiation dose.Similar to autophagy, ferroptosis may protect tumors.Ma et al. reported that IR activates the AdipoR1/NRF2 signaling pathway to inhibit ferroptosis, thereby reducing the radioresistance of liver cancer cells [141].However, no relevant evidence exists in the field of radiation injury, and further research is needed to confirm this.Research on IR-induced necroptosis in the context of radiation injury is scarce, with poorly understood underlying mechanisms.
In most studies, identifying pyroptosis, ferroptosis, and necroptosis primarily relies on changes in classic targets, with limited research on the specific upstream and downstream mechanisms.Evidence suggests potential intrinsic connections between different cell death pathways [142,143].While radiation can induce multiple forms of cell death simultaneously [144,145], research on the transformation between these death modes is lacking.The upregulation of the NFR2 induced by radiation is implicated in the occurrence and progression of ferroptosis and necroptosis [95,102,146].Research on cell death is continuously evolving.Whether other forms of cell death, such as cuproptosis, alkaliptosis, parthanatos, oxeiptosis, and disulfidptosis, are involved in the occurrence of radiation damage requires additional evidence.
Currently, there are limited clinically effective radiation protection drugs.Inhibitors related to pyroptosis, ferroptosis, and necroptosis have been summarized, and preclinical studies have shown their potential in attenuating radiation injury.However, further clinical trials are necessary for more credible evidence.Wang et al. summarized the biomarkers of pyroptosis, ferroptosis, and necroptosis, but most of these studies rely on single characteristic molecules to identify specific forms of cell death, which may not fully reflect their actual impact in RT [126].Detecting specific death modes during treatment remains a concern.Drugs with cell death inhibitory effects, such as tetrahydrobiopterin [147], enalapril [138], captopril [135], fenofibrate [148], etc., are being investigated in clinical trials related to radiation injury.However, it is yet to be proven whether the efficacy of these drugs, developed to treat radiation injury, is primarily attributed to inhibiting cell death.The clinical translation of therapies based on cell death modulation is still in the developmental stage.
In clinical settings, radiation injury occurs when normal tissues receive radiation doses intended for non-tumor target areas.However, preclinical models often employ high radiation doses to induce damage, not accurately reflecting the clinical situation.Most studies only mention the total exposure dose in the dosimetry section, lacking information on exposure dose rate, fractionation, and exposure time intervals.To facilitate further progress in research, we provide detailed evidence on the establishment of animal models and radiation injury mechanisms.Some studies may have conflicting dosimetry [109,149], likely attributed to heterogeneity across studies.Furthermore, due to species differences, there is still a considerable gap before research findings from preclinical models can be translated into clinical practice.FLASH, proton, and heavy ion RT are innovative RT methods aiming to spare normal tissues while effectively targeting tumor cells.Studies have shown that compared to conventional therapy, FLASH-RT induces lower levels of pyroptosis and intestinal injury [110].Investigating pyroptosis, ferroptosis and necroptosis can unveil the protective mechanisms of FLASH, proton, and heavy ion RT on normal tissues.
This article provides a comprehensive review of the involvement of pyroptosis, ferroptosis, and necroptosis in radiation injury generation and their underlying mechanisms.A particular emphasis is placed on the relevant dosimetry, serving as an important reference for future studies.The use of cell death inhibitors specific to these pathways holds promise for providing radioprotective effects.Further research in this field will contribute to establishing a robust theoretical foundation for effectively harnessing pyroptosis, ferroptosis, and necroptosis to mitigate radiation injury.